The long term goal of this research is to elucidate the physical mechanism of voltage-dependent activation in K+ channels by identifying structural interactions in the voltage sensor and characterizing their rearrangements during activation. Shaker and ether a go-go (eag) K+ channels will be expressed in Xenopus oocytes for electrophysiological, biochemical, and spectroscopic analysis. Unlike Shaker, eag activation is dramatically modulated by extracellular Mg2+. To obtain unique insights into voltage sensor, in voltage-dependent transitions during activation will be investigated. The specific aims of the proposal are: 1) To test the hypothesis that eag-specific acidic residues in S2 and S3 compose the Mg2+ binding site. 2) To test the hypothesis that the Mg2+ binding site in eag represents a general structural constraint in other K+ channels, including HERG and Shaker. 3) To identify structural constraints in the Shaker voltage sensor. This aim concludes work in the previous period. 4) To test the feasibility of site-directed fluorescent labeling in eag, and then use this approach to test the hypothesis that the S2 segment participates in rate-limiting, Mg2+-sensitive, conformational changes at hyperpolarized potentials during eag activation. Dr. F. Bezanilla of UCLA will collaborate in these experiments. This proposal describes basic research aimed at understanding the structure and function of voltage-dependent ion channels. The research is likely to have significant health relevance because ion channels play essential biological roles in the brain, heart, and skeletal muscle. The research may also contribute to our arrhythmias and neurological seizures. Among K+ channels, eag homologues, which are widely expressed in the brain and heart, are uniquely regulated by Mg2+, and thus may underlie some of these effects.